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Star Republic: Guide for Biologists

Common sources of cloning contaminations

  • 1. Incomplete digestion of vectors or inserts. Make sure digestion is complete, and if necessary gel purify digested products. Restriction enzymes loss activity when placed at room temperature. Don't assume the digestion will always be complete if excess enzymes are added.
  • 2. Star activity. Excess enzyme or prolonged digestion may cause star activity, i.e., unwanted digestions.
  • 3. Self-ligation of vectors or inserts. Vectors are frequently dephosphorylated by SAP or CIP to reduce self-ligation.
  • 4. The amount of digested vector or insert is too small. The relative level of contamination will be high.
  • 5. Vector to insert ratio. Commonly used ratio is vector:insert 1:3.
  • 6. Design. The construct design may have a mistake.
  • 7. Gel purification. Gel is in contact with UV box, scapula, gel tank, running buffer, gloves, etc. Avoid contamination by cleaning gel tank, change to fresh running buffer, use clean scapula, wrap the gel in a clean plastic when working on the UV box, and change to new gloves.
  • 8. Common reagents. When taking solutions from stock solutions such as ddH20, enzymes, or reaction buffers, always change to a clean sterile tip. If possible, sterilize all solutions, tips, and microcentrifuge tubes etc. by autoclaving. Bottles used for solutions can also be contaminated if not washed thoroughly.
  • 9. Competent cells. Check if competent cells (without transformation) will grow on plates with antibiotics.
  • 10. Instability of constructs. AT-rich regions, toxic sequences, and repeats may cause plasmid recombination.